Experimental set up
The three species float occupy a similar ecological niche. They float on the water surface and are very similar in terms of their morphology. In particular, non-gibbous L. gibba and L. minorfronds cannot be distinguished by eye but the differentiation betweenL. minor and L. turionifera can be equally challenging (Senevirathna et al., 2021). Therefore, we separated the species using a floating ring (Figure S4). Thus, we could overcome previous limitations (Clatworthy & Harper, 1962) and investigate the competition between these closely related species.
For each treatment, 18 250ml bottles were filled with 100ml of Hoagland’s E Medium. To create the Zn pollution gradient, ZnSO4 * 7H2O (Alfa Aesar, Thermofisher, Germany) was added (2 mg/L, 8 mg/L, 50 mg/L). Elemental Zn accounts for 65.38 g/mol (22.7 %) of the ZnSO4 * 7H2O compound, the final concentration of the metal Zn was consequently 0.45 mg/L Zn, 1.82 mg/L Zn and 11.35 mg/L Zn. The medium was autoclaved before the experiment.
We grew every treatment in three replicates. In total, we had 72 bottles and 108 individual populations (36 growing in the isolated setting and 72 growing in the mixed setting). At the beginning of the experiment, we added 5-7 fronds to the 250ml bottles according to the study design (Figure 1). In the isolated setting, the 5-7 fronds were added to the inner area (inside the plastic ring). No fronds were added to the outer area. In the mixed setting, 5-7 fronds from one species were added to the inner area and 5-7 fronds from a second species were added to the outer area. The inside/outside position of the two respective species were kept for the replica but changed depending on the concentration, so that the position was the same for 0 mg/L Zn and 1.82 mg/L Zn, and for 0.45 mg/L Zn and 11.35 mg/L Zn. There was no significant effect of position (inside vs. outside area, Figure S1). The bottles were kept in an incubator (Figure S4) at 20 °C with a light regime of 16/8h light/dark for 17 days. During the experiment, the species competed for the same nutrients in an uncrowded culture, but there was no competition for light.
The number of fronds were counted 8 times over the course of 17 days (Figure S2). Completely white (dead) fronds were not included in the total number of fronds. During the experiment, the species got mixed in a small number of bottles. Due to their morphological similarity, the species could not be reliably distinguished any more and consequently any data from these bottles post the mixing event were excluded.